1. Field of the Invention
The present invention relates to a chemiluminescence measuring apparatus for detecting chemiluminescence and bioluminescence of a substance which is contained in a liquid specimen, with high sensitivity and accuracy. The present invention also relates to a microbe count function for detecting ATP chemiluminescence of microbes to control a contamination level.
2. Background Art
Microbe monitoring for environmental control of medicinal chemical manufacturing plants and the like involves counting of airborne microorganisms, falling microorganisms, and adherent microorganisms. The method for counting is defined by International Organization for Standardization ISO 14698-1, and the cleanliness measured by the method is expressed by grades. Airborne microorganisms are generally measured by methods using the gravity-drop of airborne microorganisms or the airborne microorganism sampler which sucks a certain amount of air as disclosed in JP Patent Publication (Kokai) No. 2002-153259 A. In the methods, usually, microorganisms are collected on agar plates for a certain period of time to culture, and the cleanliness of the environment is evaluated by the number of colonies developed after the culture. The agar plates are generally cultured in a temperature controlled incubator for a few days, and the numbers of developed colonies are visually counted. The numbers of colonies in the agar plates are averaged to obtain the mean number of airborne microorganisms. In a manufacturing facility of aseptic medical products or Cell Processing Center (CPC) for producing cells which include clean rooms with a high level of cleanliness, among the above described grades, a grade A or B should be consistently maintained and controlled. These grades A and B correspond to the number of particulates in the air of 3,530/m3 or less, and the number of microorganisms of 10 CFU (colony forming unit)/m3 or less.
Meanwhile, for a contamination control of food, river, sewage disposal, and the like, a method is used in which luciferase and luciferin are added as chemiluminescence reagents to ATP (adenosine triphosphate) in microbes to measure the bioluminescence generated from the ATP. The obtained luminescence intensity is calculated into the number of microbes as disclosed in JP Patent Publication (Kokai) No. 2000-314738 A and JP Patent Publication (Kokai) No. 7-83831 A (1995) for example, so that the contamination level can be controlled. According to FIG. 6 of Nippon Nogeikagaku Kaishi Vol. 78, No. 7, pp. 630-635, 2004, the quantification limit of Escherichia coli is about 100 CFU/mL (with a reproducibility of 10.2% after N experiments where N=10). At the same time, according to FIG. 2 included in the instruction of a reagent kit which is shown in FIG. 6 of the above non-patent document, the quantification limit of Escherichia coli is about 200 CFU/mL, and the specimen solution supplied for measuring chemiluminescence is about 0.1 mL, which indicates that the quantification limit of Escherichia coli of this method is considered to be about 10 to 20 CFU.
ATP chemiluminescence assay can be applied to the measurement of airborne microorganisms. That is, microbes and dusts are collected onto an agar which is held in a Petri dish by an airborne microorganism sampler, and after an addition of a development solution, the number of airborne microorganisms included in the collected sample is calculated using ATP chemiluminescence, so that the number of living microbe, the microorganisms that are alive, is counted.
ATP chemiluminescence assay is conducted using a reagent kit provided by a certain manufacturer in accordance with the measurement procedure which is as follows:
(A) an ATP eliminating reagent is dispensed in a sample solution tube so as to eliminate killed microbes and ATP other than living microbes;
(B) another ATP eliminating reagent is dispensed in the sample solution tube so as to extract ATP from the living microbes;
(C) a chemiluminescence reagent is dispensed in the sample solution tube; and
(D) move the tube which contains the chemiluminescence reagent and the mixed solutions to a black box of an apparatus for measuring the amount of chemiluminescence.
In such an ATP chemiluminescence assay, in order to measure ATP chemiluminescence with high sensitivity and accuracy, the use of a detector with high sensitivity and the achievement of a high concentration by an optical arrangement of the detector and a chemiluminescence reaction field are the important factors. Moreover, a light shielding set up for constraining the entrance of so-called stray light as much as possible is another important factor because the entrance of stray light which comes from the exterior of the apparatus or chemiluminescent substance into the apparatus decreases the accuracy of chemiluminescence measurement.
First, as to the detector with high sensitivity, conventionally, photomultipliers have been used as a photodetector of a microbe count apparatus which has a luminometer for ATP measurement or a luminometer using ATP chemiluminescence. When a higher sensitivity is needed, a single photon counting method (photon counting method) for digitally processing the signals from a photomultiplier is used.
Next, as to the optical arrangement, because a chemiluminescence intensity is decreased inversely by the distance square from a chemiluminescence emitting point, it is considered to be effective to place a specimen container having a chemiluminescence substance therein closer to a sensitive area. Also, the chemiluminescence from the luminous point is scattered in a sphere, the optical arrangement which allows an effective collection of the chemiluminescence to a sensitive are is important. A chemiluminescence collection efficiency is often defined using a solid angle, and according to the definition, a sensitive area which is closer to a container and larger relative to the luminous area is important to achieve a higher sensitivity. Also, specular members which surround a container holder are effective to cause chemiluminescence to be forcibly reflected at the specular surfaces to be introduced to the sensitive area.
Finally, to address stray light (to prevent an entrance of stray light), generally, a photodetector and a specimen container are covered with a light-shielding box, that is, the entire apparatus for chemiluminescent assay and detection is completely covered with a shielding body to block stray light.
However, a microbe count apparatus which uses ATP chemiluminescence has a solution control section therein for dispensing and collection of a solution in addition to a photodetector, which increases an area of the apparatus to be shielded, and also the material may includes a luminous element. This makes it difficult to block an entrance of stray light.
Thus, it is effective to partially shield an apparatus for chemiluminescent assay and detection from light, and JP Patent Publication (Kokai) No. 7-83831 A (1995) discloses a case where a luminometer is used to achieve the partial shielding. Generally, an openable/closable shutter unit is placed in front of a sensitive area of a photodetector to shield light (hereinafter, referred to as “double light shielding type”). The unit prevents an entrance of light to the chemiluminescence detecting means just prior to a sensitive area. Therefore, no stray light hits a light-receiving element, which prevents degradation and variation in dark currents due to an accumulation of lights caused by the stray light.